I. Field of the Invention
The present invention relates to the field of compositions, analysis and quantification of apolipoproteins in biological and clinical samples. More particularly, this invention relates to methods, techniques, and protocols for the fingerprinting, profiling, determining, and/or quantifying of apolipoproteins present in samples of human biological matrices, such as, for example and without limitation, plasma, urine, serum and lipoprotein fractions.
II. Description of the Related Art
It is well accepted that apolipoproteins, the protein components of lipoproteins, are able to solubilize hydrophobic lipids and facilitate cell targeting and transport. These components, synthesized in the liver and intestine, are essential for maintaining the integrity of lipoprotein particles, serving as cofactors for enzymes that act on lipoproteins, and facilitating receptor-mediated interactions that remove lipids from circulation.
There are several groups of apolipoproteins: A (Apo A), B (Apo B), C (Apo C) and E (Apo E). Each of the three groups A, B and C consists of two or more distinct proteins. These are Apo A1, Apo A11, Apo AIV, and AV for Apo A; Apo B100 and Apo B48 for Apo B; and Apo CI, Apo Cli, Apo CIII, and Apo CIV for Apo C. Apo CI, CIII, CIV and Apo E each consist of two or more isoforms. The apolipoproteins have various roles in disease and in health.
Apolipoprotein CIII is a 79 amino acid protein that exists in humans as three isoforms differing in the glycosylation at Threonine-74. CIII-0 isoform is the final product of sialidase enzyme reactions, has an absence of sialic acid, galactose and galactose amine residues, and accounts for 14% of the total isoforms. CIII-0 isoforms are inhibitors of very low density lipoproteins (VLDL) binding to the lipolysis stimulated receptor, which is an important route of clearance of high triglyceride lipoproteins in plasma. CIII-0 isoform has the lowest affinity to VLDL. CIII-1 isoform has 1 mole of sialic and counts for 51% of total isoforms. CIII-2 isoform is the initial form of CIII synthesized and secreted in the liver, has 2 mole of sialic acid, and accounts for 35% of total isoforms. CIII-2 isoform has a higher affinity to VLDL and is a poorer inhibitor of VLDL binding to the lipolysis stimulated receptor.
Current methods to identify and quantitate CIII isoforms involve isolation of lipoprotein fractions from plasma, delipidation of this fraction, and purification of the CIII from the water-soluble fraction of apolipoproteins. Although the different purified isoforms can all be detected by isoelectric focusing gel electrophoresis, mass spectrometry, and fluorescence and absorbance spectroscopy, no individualization, differentiation, or relative quantification of a mixture of isoforms is provided by or within such results. Separation of the isoforms must be accomplished first, such as by ion exchange chromatography, if specific detection information regarding a particular isoform is desired. That is, current methods are disadvantageous in that they are tedious in isolation of CIII from plasma, having only a 60-80% protein recovery, and in that current methods for clinical diagnostics for CIII in plasma, such as ELISA and immunoturbidimetric assays, only can detect total CIII.
However, CIII isoforms are clinically significant for several reasons. The levels of the isoforms change with the level of glucose control in diabetic patients.1 For example, high HbA1C directly correlates to high CIII-0 levels.2 Hypertriglyceridemic subjects have an increased proportion of CIII as the CIII-2 isoform in VLDL. CIII-2 levels increase in females subjected to severe caloric restriction despite normal total CIII levels. The variation in CIII-2 positively correlates to changes in VLDL triglycerides while the variation of CIII-1 inversely correlates. Because of the limitations in the existing techniques with respect to the accurate quantitation and detection of the CIII isoforms, the role of these isoforms in various metabolic processes, although of great importance in understanding lipid metabolism, is still subject to controversy. 1Sundsten, T., Ostenson, Claes-Goran, Bergsten, P., Diabetes/Metabolism Research Reviews, 24(2):148-54, 2008 February, Serum Protein Patterns in Newly Diagnosed Type 2 Diabetes Mellitus—Influence of Diabetic Environment and Family History of Diabetes.2Florez, H., Mendez, A., Casanova-Romero, P., Larreal-Urdaneta, C., Castillo-Florez, S., Lee, D., and Goldberg, Ronald., Atherosclerosis, 188(1):134-41, 2006, September, Increased Apolipoprotein C-III Levels Associated with Insulin Resistance Contributed to Dyslipidemia in Normoglycemic and Diabetic subjects from a Triethnic Population.
The literature or prior art report various characterizations and correlations between various apolipoproteins and diseases. For example, Apo D is a multi-ligand, multifunctional transporter and is known to accumulate in a specific site of regenerating peripheral nerves in Alzheimer's disease.3 Further, Apo J so far has been reportedly implicated in several diverse physiological processes, such as sperm maturation, lipid transportation, complement inhibition, tissue remodeling, membrane recycling, cell-cell and cell-substratum interactions, stabilization of stressed proteins in a folding-competent state, and promotion or inhibition of apoptosis. Also, Apo H is known to bind tightly to negatively charged surfaces and to inhibit the activation of the intrinsic pathway of blood coagulation and the prothrombinase activity of activated platelets by covering the negatively charged surfaces necessary for both activities. Apo F associates with LDL and inhibits cholesterol ester transfer protein (CETP) activity, and appears to be an important regulator of cholesterol transport. Apo F associates to a lesser degree with VLDL, Apo A1 and Apo A11. Apo M was proposed to be involved in lipid transport.4 Apo CIV is a 14.5 kD size apolipoprotein in the same locus as CI and CII, yet no function appears to be reported in the literature. 3Shuvaev, et al., Neurobiology of Aging, 22(3):397-402, 2001 May-June. Increased protein glycation in cerebrospinal fluid of Alzheimer's disease; Christen, Y. American Journal of Clinical Nutrition, 71(2):621S-629S, 2000 February. Oxidative stress and Alzheimer disease; Sasaki, et al., American Journal of Pathology, 153(4):1149-55, 1988 October. Advanced glycation end products in Alzheimer's disease and other neurodegenerative diseases.4Feingold, et al., Atherosclerosis, 199(1):19-26, 2008 July. Infection and inflammation decrease apolipoprotein M expression; Huang, et al. Medical Hypotheses, 69(1):136-40, 2007. Apolipoprotein M likely extends its anti-atherogenesis via anti-inflammation; Dahlback, et al. Current Opinion in Lipidology, 17(3):291-5, 2006 Jun. Apolipoprotein M—a novel player in high-density lipoprotein metabolism and atherosclerosis.
Further, based on epidemiological correlation between cardiovascular disease and cholesterol levels, clinicians have long measured and standardized the measurement of cholesterol levels to assess risks of heart disease. Lipoprotein particles (LDL and HDL) and the cholesterol associated with them also have been used in the assessment of cardiovascular risks.5 Many research studies have been conducted to relate health effects to lipoprotein particle sizes and densities, but the conclusions from these studies have not been consistent. 5Chen, et al., International Journal of Urology, 12(10):886-91,2005 October. Antiandrogenic therapy can cause coronary arterial disease; Florez, et a., Atherosclerosis, 188(1):134-41, 2006, September, Increased Apolipoprotein C-III Levels Associated with Insulin Resistance Contribute to Dyslipidemia in Normoglycemic and Diabetic subjects from a Triethnic Population; Scheffer, et al. Clinical Chemistry 54(8):1325-30, 2008 August. Increased plasma apolipoprotein C-III concentration independently predicts cardiovascular mortality: the Hoorn Study; Gervaise, et al. Diabetologia, 43(6):703-8, 2000 June. Triglycerides, apo C3 and Lp B:C3 and cardiovascular risk in type II diabetes; Huang, et al. Medical Hypotheses, 69(1): 136-40, 2007. Apolipoprotein M likely extends its anti-atherogenesis via anti-inflammation; Dahlback, et al. Current Opinion in Lipidology, 17(3):291-5, 2006 June. Apolipoprotein M-a novel player in high-density lipoprotein metabolism and atherosclerosis.
Over the last few years, there has been considerable evidence that apolipoprotein levels are associated with a variety of conditions, and recently the National Heart, Lung, and Blood Institute (NHLBI), in a recent meeting with the Centers for Disease Control and Prevention, recommended that apolipoprotein B measurements be included for standardization in the near future. Apolipoproteins have been associated with cardiovascular disease, diabetes, stroke, obesity, Alzheimer's, HIV, and other diseases.6 Given the primary role of apolipoproteins in the transport and metabolism of lipids, these associations are not surprising. The difficulty of purifying, detecting, and quantifying apolipoproteins, however, has not made it easy to conduct investigations between levels of these compounds and health effects. 6Allard, et al. Proteomics. 4(8):2242-51,2004 August. ApoC-1 and ApoC-III as potential plasmatic markers to distinguish between ischemic and hemorrhagic stroke; Sierra-Johnson, et al., European Heart Journal, 28(21):2637-43, 2007 November. ApoB/apoA-1: an independent predictor of insulin resistance in US non-diabetic subjects; Florez, et al., Atherosclerosis, 188(1):134-41, 2006 September. Increased apolipoprotein C-III levels associated with insulin resistance contribute to dyslipidemia in normoglycemic and diabetic subjects from a triethnic population; Malik, et al. Clinical Cancer Research, 11(3):1073-85, 2005 Feb. 1. Serum levels of an isoform of apolipoprotein A-II as a potential marker for prostate cancer; Chen, et al., International Journal of Urology, 12(10):886-91, 2005 October. Antiandrogenic therapy can cause coronary arterial disease; Sundsten, et al., Diabetes/Metabolism Research Reviews, 24(2):148-54, 2008 February. Serum protein patterns in newly diagnosed type 2 diabetes mellitus-influence of diabetic environment and family history of diabetes; Alborn, et al. Clinica Chimica Acta, 378(1-2):154-8, 2007 Mar. Relationship of apolipoprotein A5 and apolipoprotein C3 levels to serum triglycerides in patients with type 2 diabetes; Florez, et a, Atherosclerosis, 188(1):134-41, 2006, September, Increased Apolipoprotein C-Ill Levels Associated with Insulin Resistance Contribute to Dyslipidemia in Normoglycemic and Diabetic subjects from a Triethnic Population; Dallinga-Thie, et al. Diabetoloia 49(7):1505-11, 2006 July. Plasma apolipoprotein A5 and triglycerides in type 2 diabetes; Davidsson, et al., Journal of Lipid Research, 46(9):1999-2006, 2005 September. A proteomic study of the apolipoproteins in LDL subclasses in patients with the metabolic syndrome and type 2 diabetes; Rimland, et al., Journal of Acquired Immune Deficiency Syndromes: JAIDS, 42(3):307-13, 2006 July. Antiretroviral therapy in HIV-positive women is associated with increased apolipoproteins and total cholesterol; Feingold, et al., Atherosclerosis, 199(1):19-26, 2008 July Infection and inflammation decrease apolipoprotein M expression; Shuvaev, et al., Neurobiology of Aging, 22(3):397-402, 2001 May-June. Increased protein glycation in cerebrospinal fluid of Alzheimer's disease; Christen, Y. American Journal of Clinical Nutrition, 71(2):621S-629S, 2000 February. Oxidative stress and Alzheimer disease; Sasaki, et al., American Journal of Pathology 153(4):1149-55, 1988 October. Advanced glycation end products in Alzheimer's disease and other neurodegenerative diseases; Rosenberg, et al., Molecular & Cellular Biology, 22(6):1893-902, 2002 March. Apolipoptrotein J/clustering prevents a progressive glomerulopathy of aging; Scheffer, et al. Clinical Chemistry, 54(8): 1325-30, 2008 August Increased plasma apolipoprotein C-III concentration independently predicts cardiovascular mortality: the Hoorn Study; Gervaise, et al. Diabetologia, 43(6):703-8, 2000 June Triglycerides, apo C3 and Lp B:C3 and cardiovascular risk in type II diabetes; Ordonez, et al. Histology & Histopathology, 21(4):361-6, 2006 April Apolipoprotein D expression in substantia nigra of Parkinson disease; Tozuka, et al. Annals of Clinical & Laboratory Science, 27(5):351-7, 1997 September-October Characterization of hypertriglyceridemia induced by L-asparaginase therapy for acute lymphoblastic leukemia and malignant lymphoma; Favrot, et al. Biomedicine & Pharmacotherapy, 38(1):55-9, 1984. Study of blood lipids in 30 children with malignant hematological disease or carcinoma; Huang, et al. Medical Hypotheses, 69(1):136-40, 2007. Apolipoprotein M likely extends its anti-atherogenesis via anti-inflammation; Dahlback, et al. Current Opinion in Lipidology, 17(3):291-5, 2006 June Apolipoprotein M-a novel player in high-density lipoprotein metabolism and atherosclerosis.
Using prior art methodology, the quantification and measurement of apolipoprotein currently requires the step of separation of lipoprotein particles from serum by analytical or sequential ultracentrifugation, column chromatography, electrophoresis, or precipitation. These traditional techniques currently are too expensive and time consuming for routine clinical use. Another useful technique is high performance liquid chromatography, which is faster but much more complex and expensive. Other techniques presently used for measurement of Apo A and B content include enzyme immunoassay (ELISA), radioimmunoassay, fluorescence immunoassay, radial immunodiffusion, nephelometry, turbidimetry and electroimmunoassay. Recently, surface-enhanced laser desorption ionization mass spectrometry (SELDI or SELDITOF-MS, with TOF meaning time of flight and MS meaning mass spectrometry) has offered some new options for measuring apolipoproteins in plasma, serum, and lipoprotein fractions. However, each known technique is disadvantageous at least in that a single apolipoprotein must be targeted for quantification and measurement, thus requiring a plurality of lengthy, complex, and expensive techniques to be performed if information regarding a plurality of apolipoproteins is desired. Further, profiles of total plasma or serum require elaborate clustering and pattern recognition techniques to detect differences in samples of normal and healthy individuals.
Accordingly, there is always a need for accurate, rapid, reproducible assays for the separation, identification, and quantification of apolipoproteins. There is a need for improved techniques for the fingerprinting, profiling, determining, and/or quantifying of apolipoproteins present in biological matrices, such as samples of human plasma, serum and lipoprotein fractions, urine, or the like, whereby the phenotype of a particular individual human may be characterized in lieu of or in addition to the genotype. It is to these needs among others that the present invention is directed.